Method for diagnosing cancers

ABSTRACT

A method of diagnosing and/or monitoring the development of cancer by analyzing the deoxyribonucleic acid (DNA) in blood plasma, and particularly by detecting any gene alterations in cancer cell DNA, e.g. oncogene mutations or deletions, tumour suppressor gene mutations or deletions, or microsatellite alterations.

The present invention is concerned with a method for diagnosing and/ormonitoring the evolution of various types of cancers after achemotherapeutic treatment or after surgery.

It is well known that the diagnosis and the monitoring of the evolutionof cancers is carried out, in addition to the observation and the directexamination of the tumors, by the analysis of biopsy samples, or in thecase of blood cancers, an examination of the bone marrow. This implieseither a surgical intervention, or an invasive operation of the biopsytype or further a bone medulla aspiration using a needle. Actually, inaddition to the unpleasant if not dangerous nature of such methods tothe patient, it was found that they could furthermore lack accuracy. Inthe case of certain leukemic diseases for example, the analysis of thesample of bone marrow taken has not made it possible to identify all themalignant clone varieties.

The purpose of this invention is hence to provide a method fordiagnosing cancers which, on the one hand, is more accurate and morereliable and which, on the other hand, is easier to carry out withoutthe need of resorting to invasive tests on the patients.

The method for diagnosing and/or monitoring the evolution of cancers,object of the invention and aimed at achieving the above purposes,includes the analysis of the deoxyribonucleic acid (DNA) contained inthe blood plasma.

In fact, it has now been possible to demonstrate that patients sufferingfrom different cancer diseases show increased amounts of DNA in theblood plasma. The method of diagnosis according to the invention ishence based on the detection of gene mutations in this plasma DNA, theblood plasma being a human material much more easily accessible thatbiopsy samples of tumors for example. Thus, oncogene mutations arefrequently evidenced in numerous types of malignant tumors and, amongstthem the mutations of the ras gene are particularly meaningful. However,the method can be applied to any gene modification of the DNA of thecarcinomatous cells, such as mutations and deletions of the genes ras,APC, DCC, P53, etc of any oncogene or anti-oncogene gene (tumorsuppressing gene) or furthermore modifications in the microsatellites.It has even been found that different mutations of the ras gene detectedin the DNA of the blood plasma could be absent in the DNA of peripheralblood cells or in the case of certain patients suffering from leukemiain the DNA of bone marrow, which tends to confirm the greaterreliability of the method according to the invention by comparison toknown diagnostic methods.

Generally, the method of diagnosis according to the invention consistsin extracting the DNA from blood plasma, in purifying and amplifyingthis DNA, and thereafter in determining gene mutations or deletionstherein, in principle in a comparative test, with the blood plasma of aperson presumed to be ill and that of another person in good health, asreferences.

The scope of the present invention extends to any technique forextracting, purifying or amplifying blood plasma DNA; also, any methodappropriate for determining gene mutations can be used.

The diagnostic method according to the invention will now be describedin more detail with reference to the two following examples:

EXAMPLE 1

Diagnosis of the cancer of the colon by detection of mutations of theK-ras gene.

In this first application of the method according to the invention, usewas made of the determination of mutations in the codon 12 of the K-rasgene contained in the adenocarcinomata of the colon. These mutationsoccur generally upon transition from stage I adenoma to stage IIadenoma, before the deletion or the mutation of gene P53, i. e. at arelatively early stage of the development of the tumor.

Blood samples (20-30 ml) were taken from 15 patients with a colo-rectaladenocarcinoma at different stages of development and heparinized, saidpatients having received at that time no anticancer drug. Thirteen ofthe 15 patients underwent subsequently surgery to remove the tumor;also, a total of approximately 400 ml of blood were taken from healthypersons, in order to isolate their plasma DNA.

The DNA was extracted from the tumors and from blood cells using wellknown conventional techniques.

As to the extraction of the DNA from blood plasma, it can be carried outas follows: the plasma is first subjected to a treatment using phenol,ether and chloroform. After dialysis against a SSC buffer (sodiumchloride 0.15 M, trisodium citrate 0.015 M), the product is passedthrough a concanavallin A-Sepharose® column in order to eliminate thepolysaccharides and then centrifuged in a Cs₂ SO₄ gradient.

The DNA thus extracted and purified (10 to 100 ng) is then subjected toan amplification by PCR of the first exon of the K-ras gene in a volumeof 100 μl.

The amplimers were

5'-GACTGAATATAAACTTGTGGTAGT-3' (SEQ ID NO:1) and

5'-CTATTGTTGGATCATATTCGTCC-3' (SEQ ID NO:2)

The amplifications were carried out in a buffer containing 50 mM of KCl,10 mM of Tris-NaCl pH 0.3, 200 mM of each nucleotide, 1.8 mM of MgCl₂,0.2 μM of each precursor and 2.5 units of "AmpliTaq"® DNA polymerase. 35cycles were carried out for the DNA of the tumors and of the blood cellsand 45 cycles for the DNA of the plasma (94° C. during 1 min, 59° C.during 1.5 min, 72° C. during 1 min, the last cycle being extended to 7min at 72° C.).

Concerning the detection of the mutations, it can be carried out by anyknown appropriate method. In the present example, it was carried out bytwo different techniques for each sample tested.

(a) Hybridization of the PCR products with oligonucleotide probes whichare mutation specific (according to Verlaan de Vries et al, Gene 50,313-320, 1986):

The PCR products were placed in equal amounts on "Zeta-probe"® membranes(Bio-Rad, Hercules, Calif.) and hybridized with oligonucleotidesspecific for wild-type or mutant K-ras. The oligonucleotides werelabelled with 32-P ddATP (Amersham GB). In order to separate the perfecthybrids from mismatches, the final washing of the membranes was carriedout in a solution containing tetramethylammonium chloride 3 M, 50 mM ofTris-HCl at pH 8.0, 0.2 mM of EDTA and 0.1% of SDS, at 58° C. during 1hour.

(b) Amplification by PCR with amplimers specific of point mutations oramplification by PCR for specific alleles (PASA) (according to Sommer etal, Biotechnique 12, 82-87, 1992):

In this method, which is more sensitive, the DNA is subjected to anamplification by PCR with amplimers complementary to the normal GLYsequences or the mutated ALA, VAL, SER, ASP or CYS sequences. Theseamplimers which are specific for the mutation have 3' terminationscomplementary to the mutations at the specific point. The enzyme Taq Ipolymerase (Perkin-Elmer Cetus, SWI) has no exonuclease activity at the3' terminus and is therefore unable to amplify the DNA if the mismatchof a single base is located at the 3' terminus of the amplimer.

Each PCR was carried out in a volume of 40 μl of a solution containing50 mM of KCl, 10 mM of Tris-HCl at pH 8.3, 2 mM of each nucleotide, 0.7mM of MgCl₂, 0.2 mM of each precursor and 1 unit of "AmpliTaq"® DNApolymerase. Thirty five cycles were carried out (94° C. during 1 min,tempering at 55-62° C. during 2 min, extension at 72° C. during 1 min).The last cycle was extended to 7 min at 72° C. Each reaction wasinitiated by the "hot-start" technique. The amplimers used were thefollowing:

5'-ACTTGTGGTAGTTGGAGCTGG-3' (SEQ ID NO:3) for the wild-type K-ras(renaturation at 55° C.), 5'-ACTTGTGGTAGTTGGAGCTGC-3' (SEQ ID NO:4) forthe ALA 12 mutant (renaturation at 62° C.), 5'-ACTTGTGGTAGTTGGAGCTGT-3'(SEQ ID NO:5) for the VAL 12 mutant (renaturation at 61° C.),5'-ACTTGTGGTAGTTGGAGCTA-3' (SEQ ID NO:6) for the mutant SER 12(renaturation at 59° C.), 5'-ACTTGTGGTAGTTGGAGCTGA-3' (SEQ ID NO:7) forthe mutant ASP 12 (renaturation at 60° C.), 5'-ACTTGTGGTAGTTGGAGCTT-3'(SEQ ID NO:8) for the CYS 12 mutant (renaturation at 59° C.) and in eachcase the anti sense amplimer 5'-CTATTGTTGGATCATATTCGTCC-3' (SEQ IDNO:2).

After amplification, the reaction products were analyzed byelectrophoresis on a 0.8% polyacrylamide gel.

By using the first technique (a) described above, it was shown that itis not possible to demonstrate the same mutations in the DNA of theplasma as those detected in the DNA of the excised tumors (GLY to VAL,GYS to ALA); this technique seems to be applicable only if approximately10% at least of the total DNA exhibits a point mutation. However, theabove mutations could be identified in the plasma DNA with the secondtechnique (b) described above; it appears that this technique enablesthe identification of mutations in a sample of plasma DNA mixed with a10⁴ to 10⁵ excess of normal non-mutated DNA. On the other hand, with thesame technique, it was not possible to detect the same mutations on theDNA samples from the blood cells.

Finally, all the control samples from healthy persons showed to benegative, i. e. exhibited no mutations of the plasma DNA.

EXAMPLE 2

Diagnosis of cancers caused by myeloid disorders, using the detection ofmutations of the N-ras gene.

It is well known that a high incidence of N-ras mutations is observed inthe DNA of bone marrow of patients affected by the myelodysplasiasyndrome (MDS) or by acute myeloblastic leukemia (AML).

b 20 to 30 ml of blood were taken from ten patients affected by AML orMDS, this blood was collected on heparin and centrifuged on the "FicollHipaque" gradient (Pharmacia, SWE). Also 400 ml of blood were taken fromhealthy persons. The interphase containing mononuclear cells wascollected and used for extracting the DNA of the blood cells. The upperphase was centrifuged at 2500 G during 15 minutes and the supernatantwas used for the extraction of plasma DNA. Furthermore, several samplesof bone marrow from the same patients were taken for control analyses.

The DNA from the blood cells and from the bone marrow was isolated bytreatment with protease K (Merck GER) in the presence of SDS, followedby a phenol extraction, a precipitation with ethanol and centrifugationin a Cs₂ SO₄ gradient. The plasma DNA was extracted as described inexample 1.

The DNA (10-100 ng) was amplified in a volume of 100 μl. The amplimersused (Oncogen Science, N.Y., USA) were 5'-GACTGAGTACAAACTGGTGG-3' (SEQID NO:9) and 5'-CTCTATGGTGGGATCATATT-3' (SEQ ID NO:10) for the firstexon of the N-ras gene. The amplifications were carried out in an"Thermo-Cycler 480" (Perkin-Elmer Cetus, SWI) under the same conditionsas those of example 1. Each cycle consisted of a denaturation step at94° C. during 1 minute, a renaturation (at 51° C. for N-ras) during 1.5minutes and an extension of one minute at 72° C., with a third extensionsegment of 5 seconds per cycle. The last cycle was followed by anextension of 7 minutes at 72° C. The products of the amplification (109ng) were analyzed by electrophoresis on a 0.8% polyacrylamide gel.

The same two techniques of detection of mutations were employed as inexample 1. In the second technique (b), the amplimer used for N-ras was5'-CTGGTGGTGGTTGGAGCAGA-3' (SEQ ID NO:11) for the ASP 12 mutant,5'-GGTGGTGGTTGGAGCAGGTT-3' (SEQ ID NO:12) for the CYS 13 mutant and5'-CTCTATGGTGGGATCATATT-3' (SEQ ID NO:10) as the anti sense amplimer.

The results of the analyses obtained make it possible to confirm thatthe DNA of the diseased patients exhibited one or several mutations ofthe codon 12 (GLY to CYS or to ASP) or of the codon 13 (GLY to CYS) inthe N-ras gene, while none of these mutations could be found neither inthe DNA of the blood cells nor even in that of the bone marrow.

Thus, it is clearly apparent from the two illustrative examples abovethat the analysis of the DNA of blood plasma can provide a method fordiagnosing and monitoring the evolution of a cancerous disease, which ismore convenient, less traumatic (simply a sample of the patient's bloodneeds to be taken) and sometimes more reliable than known methods makinguse of biopsy samples.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 18    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #                24GTGG TAGT    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #                23TTCG TCC    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #21                GCTG G    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #21                GCTG C    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #21                GCTG T    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    # 20               GCTA    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #21                GCTG A    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    # 20               GCTT    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    # 20               GTGG    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    # 20               TATT    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    # 20               CAGA    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    # 20               GGTT    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    # 20               GAAC    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    # 20               CTCA    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    # 20               GTGA    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    # 20               TATA    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    #21                TACC A    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    #21                TCAG T    __________________________________________________________________________

We claim:
 1. A method for diagnosing and/or monitoring progression ofcancers, comprising extracting deoxyribonucleic acid (DNA) contained ina non-cellular blood fraction of a patient, purifying and amplifying theextracted DNA, and detecting microsatellite alterations in the amplifiedDNA, wherein the microsatellite alterations are indicative of theprogression of a cancer in said patient.
 2. A method according to claim1, further comprising detecting oncogene mutations or oncogenedeletions, or mutations or deletions of tumor suppressing genesoccurring in the DNA of cancerous cells.
 3. A method according to claim2, in which the detection is applied to any oncogene, its fullycomplementary strand or tumor suppressing gene.
 4. A method according toclaim 1, in which the extracted DNA is amplified by PCR.
 5. A methodaccording to claim 4, in which the detection of gene mutations iscarried out by hybridization of products obtained by PCR witholigonucleotide probes which hybridize specifically to the mutations. 6.A method according to claim 1, in which the detection of gene mutationsis carried out by amplification by PCR and the use of primers whichhybridize specifically to point mutations.